There are seven chapters in this thesis. Chapter I (Preface) provides a brief overview of the context of this study and the aims and objectives.
Chapter II (Literature Review) presents a review of the research on phytochemicals in fruits, vegetables, and other plant foods, with a particular focus on phenolic compounds and flavonols in Brassica species. The chemical analysis of these compounds in plants is summarised, including sample preparation, extraction, purification, and various instrumental analytical techniques. Previous research on the anti-cancer properties of these naturally occurring plant compounds is also presented, with a particular focus on cell culture models (especially the HT-29 cell line) and antiproliferative activities of the flavonol compounds that are of interest to this study and Brassica specie plant extracts.
The experimental design, and materials and methods, applied for the analyses are detailed in Chapter III (Materials and Methods).
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There are three main results and discussion chapters: chapters IV, V, and VI. Method optimisation and other preliminary studies are reported and discussed in Chapter IV. Chapter V (Identification and Quantification of Flavonols in Bok Choy Cultivars) reports and discusses the results of the chemical analysis and structure elucidation of flavonol conjugates present in three bok choy cultivars. Following on from this study, Chapter VI (Cancer Cell Studies) reports and discusses the results of the antiproliferative effects of flavonol compounds, selected fractions, and bok choy extracts on HT-29 human colorectal adenocarcinoma cells in vitro.
In Chapter VII (General Discussion, Conclusions and Future Directions), the main results are briefly discussed, the conclusions are summarised, and future directions are suggested.
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CHAPTER II
2
LITERATURE REVIEW
2.1
INTRODUCTION
Research on phytochemicals in fruits, vegetables, and other plant foods, can be divided into two main areas; the analytical chemistry of phytochemicals in plants, and their bioactivities both in vitro and in vivo. This chapter will review literature in both these areas.
Sections 2.2-2.4 provide some general information regarding phytochemicals, their classification, and health benefits, with a specific focus on the prevention and management of colorectal cancer. This is followed by further information on the particular phytochemicals of interest to this study; phenolic acids and flavonoids, which can be further subdivided into hydroxycinnamic acids and flavonols respectively. Section 2.4 then provides more detailed information concerning the plant family that is of interest to this study; Brassicaceae, and more specifically the Brassica species and the vegetable bok choy.
Sections 2.5 and 2.6 address the analytical chemistry of phytochemicals in plants, including methodologies used for extraction, purification, and instrumental analysis for quantification and structure elucidation.
Section 2.7 reviews the literature on the bioactivities and anti-cancer properties of flavonols such as antiproliferative effects on cell culture models. This is inclusive of a review on studies that investigated the three flavonols of interest in this study: quercetin, kaempferol, and isorhamnetin, as well as the effects of plant extracts, in particular Brassica species, on cancer cells in vitro. Section 2.8 provides a brief summary of the information covered in this chapter.
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2.2
PHYTOCHEMICALS
Phytochemicals are defined as biologically active non-nutrient plant compounds (‘phyto’ is from the Greek word meaning plant) and are present in all fruits, vegetables, grains, as well as other plant foods 16. Nutrients, e.g. vitamins, minerals, proteins, carbohydrates and lipids are substances that are essential for the human body; for the maintenance of life, growth, and nourishment, however, non-nutrients are not required for the body to function. The type and quantity of phytochemicals present in plant foods varies significantly from source to source. There have been more than 5,000 different phytochemicals identified to-date in an extensive variety of plant foods and it is estimated that thousands more have yet to be discovered 17.
Phytochemicals are secondary plant metabolites and many of these are produced via shikimate and chorismate biosynthesis from the essential precursor L-phenylalanine, an amino acid with an aromatic ring 18-19. The synthesis of these compounds involves a series of reactions including deamination, enzymatic conversion, and enzymatic hydroxylated reactions 20, which generates a variety of phenolic phytochemicals with one or more phenolic rings (Figure 2.1). Based on the number and pattern of phenolic rings, these phytochemicals may be relatively simple structures such as phenolic acids, which consist of a single phenolic ring, or more complex structures known as polyphenolics and tannins, which consist of two or more phenolic rings, respectively. These compounds can also conjugate with different moieties including sugars, long carbon chains, and phytosterols to form complex phenolic derivatives.
Within the plant kingdom, phytochemicals have been found to play important roles in plant metabolism in addition to being key players in defence mechanisms and disease resistance. Their major physiological function is to defend against oxidative and environmental stresses including protection against UV radiation, microbes, pathogens, and parasites 21. They can also be responsible for the colours in plants; for example, anthocyanins (Figure 2.2), are plant pigments that give fruits and vegetables their black, purple, blue, red, or orange colour. In addition to colour they also contribute to the taste in fruits and vegetables, e.g. flavanols, flavanones, and flavones can be responsible for the astringency, bitterness or spicy taste of edible plants 22-23.
7 O HO OH OH OH OH O O HO OH OH OH OH HO O HO OH OH OH O O HO OH O OH glucose shikimate L-phenylalanine p-hydroxycinnamic acid p-coumaric acid 4-coumaroyl CoA 3 x malonyl CoA glucose naringenin chalcone naringenin isoflavone (+)-catechin (flavanone) (flavanol) dihydrokaempferol dihydroquercetin delphinidin (anthocyanidin) kaempferol quercetin (flavonol) (flavonol) (anthocyanidin) chorismate pelargonidin cyanidin (anthocyanidin) OH HO OH O OH O HO OH OH OH OH O HO OH O OH O HO OH OH OH O O HO OH OH OH O OH O HO OH OH OH O HO OH OH OH OH
Figure 2.1 One of the pathways of phytochemical biosynthesis in plants. Adapted from Koh and Mitchell 20.
PAL C4H 4CL CHS CHI FHT IFS Abbreviations: ANS – anthocyanidin synthase C4H - cinnamate 4-hydroxylase CHI - chalcone isomerase CHS - chalcone synthase 4CL - p-coumarate:CoA ligase DFR – dihydroflavonol 4-reductase F3'H – flavonoid 3'-hydroxylase F3'5'H – flavonoid 3',5'-hydroxylase FHT - flavanone 3-hydroxylase FLS – flavonol synthase IFS - isoflavone synthase
PAL - phenylalanine ammonia lyase
FLS FLS DFR ANS ANS F3'H ANS F3'5'H F3'5'H
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Classification
Phytochemicals can be classified into the following five groups; carotenoids, phenolics, alkaloids, nitrogen-containing compounds, and organosulfur compounds. Figure 2.2 depicts a flow diagram of the most common classes of dietary phytochemicals. The phenolics group are the most abundant in the human diet and consist of an aromatic ring with one or more hydroxyl moieties attached. The group can be further subdivided into several classes according to their structure, i.e. the number of phenol rings they contain, as well as the structural elements that bind these rings 24-25. The main dietary classes of phenolics consist of the following five groups; phenolic acids (which comprises both hydroxybenzoic and hydroxycinnamic acids), flavonoids, stilbenes, coumarins, and tannins. The flavonoid group can be further subdivided into six major classes; flavonols, flavones, flavanols (also known as catechins), flavanones, anthocyanidins, and isoflavonoids. These compounds consist of a C6–C3–C6 structure, whereas the classes that belong to the non-flavonoid groups are classified based on the number of carbons in their structure 24. In general, phytochemicals occur naturally as glycosylated conjugates in plants and not in their aglycone form (an aglycone is the compound remaining after the glycosyl group is replaced by a hydrogen atom).
Figure 2.2
Flow diagram showing classification of dietary phytochemicals adapted from
Liu
16. Phytochemicals of interest in this study are highlighted in blue.9
Health Benefits
Increasing evidence suggests that the phytochemicals present in edible plants may also be beneficial components for human health, either individually or in combination with essential nutrients such as proteins, lipids, carbohydrates, and vitamins 17.
Many studies support the pharmacological activities of phytochemicals in humans, including anti- inflammatory, anti-allergic, antiviral, antimicrobial, anti-hepatotoxic, anti-osteoporotic, antispasmodic, antiulcer, antidiabetic, estrogenic/antiestrogenic, anxiolytic, analgesic, vasodilating, cardioprotective, neuroprotective, and anticancer activities. The most widely studied activity, however, is their antioxidant properties 26. Antioxidants are substances that inhibit the generation of oxidation-initiating free radicals. This inhibition helps to prevent or delay oxidation reactions such as lipid peroxidation, which is one of the most important actions of free radicals that leads to the damage of cell membranes and, ultimately, cell death 27. The antioxidant function of phytochemicals stems from the hydroxyl groups attached to the phenolic rings, which donate electrons to free radicals in order to stabilise them in a system. The phenolic radicals that are formed are relatively stable due to resonance occurring on the phenolic ring preventing the initiation of a new free radical chain reaction. Furthermore, the phenolic radical intermediates can react with other free radicals within the system to terminate the chain reaction. Phytochemicals can also suppress reactive oxygen and nitrogen species formation by deactivating related enzymes and chelating metal ions that produce free radicals. The antioxidant activity of a phenolic compound is directly related to its structure and the number of free hydroxyl groups on its phenolic rings 17.
In addition to their antioxidant activities, many phytochemicals alter cell signalling pathways and gene expression and therefore play important roles in preventing various chronic diseases such as cancer, diabetes and obesity, cardiovascular diseases, and lowering blood cholesterol 28. As more and more consumers become aware of the secondary health benefits of foods, it is becoming increasingly important to understand and utilise these naturally occurring, powerful antioxidant phytochemicals in health-promoting food and other products, as well as investigate the possibility of natural treatments and preventions for chronic diseases. A list of possible health benefits for some common phytochemicals is shown in Table 2.1.
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Table 2.1 Potential health benefits of some phytochemical compounds and their sources in plant foods 29, 17, 30-31, 23, 32-35, 25, 36, 8, 6.
Phytochemical Food Source Possible Health Benefits
Isoflavones Soy beans, soy milk, tofu and other legumes Lower blood pressure and increase vessel dilation, reduce the formation/progression of cancer (especially estrogen-sensitive cancers, i.e. breast, ovarian, colon, prostate), decrease total cholesterol and cardiovascular
disease, decrease triglycerides and thrombosis, reduce onset of Alzheimer’s disease and osteoporosis
Anthocyanidins and anthocyanins
(glycosylated anthocyanidins)
Berries (strawberries, blackberries, raspberries, blueberries, cranberries, bilberries), cherries, plums, pomegranates, red
grapes, red wine, red apples, red potatoes, red radishes, red onions, beans and egg plants
Improve vision, lower blood pressure, protect LDL cholesterol oxidation, display anti-inflammatory and antimicrobial activity. Act as antioxidants, inhibit nitric oxide production, induce apoptosis and therefore provide protection against cancer and heart disease. Decrease platelet aggregation,
display neuroprotective effects, may prevent obesity and diabetes Flavanones Citrus fruits (grapefruit, oranges, lemons and
limes), tomatoes and mint
Provide protection against cardiovascular disease and cancer, improve bone health and lower cholesterol. Resist neurodegenerative diseases, e.g.
Alzheimer’s disease. Flavanols and
proanthocyanidins (oligomers of
flavanols)
Red wine, grapes, cocoa, tea, cranberries, apples, plums, pears, mangoes, peaches, okra
and Swiss chard
Inhibit LDL oxidation, decrease platelet aggregation, antioxidant activity, inhibit cellular oxygenases, antimutagens, decrease tumour
initiation/promotion, induce apoptosis, inhibit pro-inflammatory responses in the arterial wall
Flavones Apples, cereals, beets, peppers, Brussels sprouts, cabbage, cauliflower, celery, parsley,
thyme, chives, kale, lettuces, spinach, tomatoes and watercress
Prevent coronary heart disease, anti-inflammatory, antimicrobial, anti-cancer activities
Flavonols Onions, apples, berries, grapes, red wine, tea, broccoli, cabbages, bok choy, kale, leeks, chives, peppers, tomatoes, spinach, Swiss
chard and watercress
Strong antioxidant activity, chelate free radical-producing metals, prevent coronary heart disease, decrease total cholesterol, decrease risk of dementia,
protect against LDL oxidation and atherosclerosis, antimutagens, decrease tumour initiation/promotion, induce apoptosis and decrease platelet
aggregation Phenolic acids
(e.g. hydroxycinnamic
acids)
Kiwi fruit, blueberries, plums, cherries, apples, pears, chicory, artichokes, carrots, lettuce,
eggplant, wheat, coffee and tea
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Phytochemical Food Source Possible Health Benefits
Stilbenes Grapes, wine, peanuts, blueberries, bilberries and cranberries
Prevent damage to blood vessels, decrease platelet aggregation/thrombosis and prevent blood clots, reduce LDL cholesterol, antioxidant activity, carcinogen detoxification, antimutagen, decrease tumour initiation/promotion Coumarins Apricots, strawberries, cinnamon and cherries Anticoagulant, antibacterial, anti-fungal, vasodilators,
anti-tumour, anti-HIV, anti-hypertension, anti-inflammatory, anti-arrhythmia, antiseptic, anti-osteoporosis and analgesic, also used to treat asthma Tannins Berries, grapes, wine, persimmons, lentils, tea
and chocolate
Powerful antioxidants and may reduce the risk of cancer Carotenoids Carrots, tomatoes, sweet potatoes, pumpkins,
squash, apricots, peaches and dark leafy green vegetables
Neutralise free radicals that cause cell damage, act as antioxidants; may reduce the risk of cancer, heart disease, age-related macular degeneration.
May enhance the immune system response Glucosinolates/
Isothiocyanates
Cruciferous vegetables such as broccoli, brussels sprouts, cabbage, cauliflower, mustard greens, turnip greens, kale and
horseradish
Neutralise free radicals that cause cell damage and protect against some cancers, decrease tumour initiation/promotion, act as antioxidants and may
increase the activity of enzymes that function in the detoxification and elimination of toxins
Sulfides and Thiols Garlic, onions, leeks, olives and scallions Decrease total cholesterol and LDL cholesterol, decrease triglycerides, decrease cholesterol and fatty acid synthesis, decrease blood pressure, decrease thrombosis, antioxidant activity, carcinogen detoxification and
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Colorectal Cancer
Colorectal cancer is the fourth most common malignant tumour worldwide 37 and the second most commonly diagnosed cancer in both men and women in Australia 38. It is estimated that one in twelve Australians will develop colorectal cancer in their lifetime, with the disease claiming the lives of almost 4,000 Australians every year 39.
Colorectal cancer, also known as bowel (colon and rectum) cancer, generally develops via a multistage process in which a series of cellular mutations occur over time. The most common type of bowel cancer is adenocarcinoma. Adenocarcinoma forms in mucus-secreting glands throughout the body, and in the case of colorectal cancer, develops in the intestinal gland cells that line the inside of the colon and/or rectum. In the early stages, the abnormal epithelial cells mutate to form benign polyps which may then undergo additional mutations and become benign adenoma and, ultimately, a malignant cancer. Later stages of colorectal cancer can spread to other sites in the body through the lymphatic or vascular system 40.
The majority of recent epidemiological, in vitro, and in vivo studies have confirmed that a diet rich in vegetables, fruits, and legumes containing naturally occurring antioxidant phytochemicals correlates with a reduced risk in cancers 17, in particular colorectal cancer. Table 2.2 lists a number of epidemiological studies on the incidence of colorectal and other related cancers and its association with the consumption of phytochemicals through fruits, vegetables, and other plant foods/beverages in the diet. Many of the in vitro and in vivo studies have shown plausible mechanisms, such as regulating specific signalling pathways and molecular markers 41, by which phytochemicals may be possible agents for the prevention and treatment of cancers. Such studies will be discussed later in this chapter and will focus primarily on phenolic compounds.
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Table 2.2 Epidemiological studies that have investigated the relationship between a diet high in fruits and vegetables containing phytochemicals and the incidence of colorectal, and other related, cancer.
Type of Study Cancer Dietary Phytochemical Reference
Case-control Gastric Carotenoids (α-carotene, β-carotene, lutein, lycopene) and flavonoids (quercetin, kaempferol, myricetin,
luteolin)
Garcia-Closas et al.42
Case-control Colorectal Flavonols, procyanidins, catechins, flavanones
Kyle et al.43 Prospective cohort Colorectal General fruit and vegetable intake Van
Duijnhoven et al.44 Prospective cohort Colorectal Total flavonoids, quercetin,
kaempferol, myricetin
Lin et al.45 Prospective cohort Colorectal Flavonols, flavones, flavanones,
flavanols, anthocyanins
Nimptsch et al.46
Case-control Colorectal Total flavonoids, anthocyanidins, flavanols, flavanones, flavones, flavonols, isoflavones, theaflavins and
thearubigins, proanthocyanidins
Xu et al.47
Meta-analysis of 23 studies (10 prospective cohort and 13 case-
control)
Stomach and colorectal
Total flavonoids, flavonols, flavanones, flavanols, anthocyanins, procyanidins,
isoflavones Woo and Kim48 Meta-analysis of 18 studies (9 prospective cohort and 9 case-
control)
Colorectal Total flavonoids, flavones, flavonols, flavanones, flavanols, anthocyanins,
isoflavones, procyanidins
He and Sun49
2.3
PHENOLICS
Phenolics are a large group of phytochemicals and are widespread in plants, especially in fruits, vegetables and associated beverages, and are of considerable interest due to their powerful antioxidant properties. These compounds consist of an aromatic ring with one or more hydroxyl groups, and their structures can range from a simple phenolic molecule to a complex high- molecular weight polymer 24. Their antioxidant ability depends on their structure, in particular the number and location of the hydroxyl groups, and other groups/moieties that may be attached to the aromatic rings. As mentioned previously, phenolics are sub-divided into five different classes; phenolic acids, flavonoids, stilbenes, coumarins, and tannins. Both the phenolic acid and flavonoid groups are of particular interest to this study and will be discussed in greater detail.
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Phenolic Acids
Phenolic acids are divided into two subgroups: hydroxybenzoic and hydroxycinnamic acids. They are found in a range of different fruits, vegetables, and grains, although the hydroxybenzoic acid content of edible plants is generally very low, except for certain red fruits, black radish, and onions. Hydroxycinnamic acids on the other hand are much more common, with the most commonly occurring compounds in edible plants being p-coumaric, caffeic, ferulic, and sinapic acids 24-25. They are secondary metabolites that are synthesised in plants as a response to environmental stress as the plant grows, for example the presence of insects and pathogens, physical lesion, and UV radiation 21. The amino acid L-phenylalanine is the pre-cursor for the synthesis of phenolic acids via the shikimate and chorismate pathway. After several reactions including deamination, enzymatic conversion, and enzymatic hydroxylation, L-phenylalanine is converted to different hydroxylbenzoic acids and other phenolic acids 17. Their generic structure is based on either a benzoic acid or cinnamic acid core (Figure 2.3). Hydroxybenzoic and hydroxycinnamic acids are formed when one of the positions on the benzoic acid/cinnamic acid aromatic ring is occupied by a hydroxyl group. The remaining four positions on the aromatic ring can be further substituted with other groups, e.g. methoxyl or hydroxyl groups. The chemical properties of the hydroxybenzoic acid and hydroxycinnamic acid derivatives differ, for example, hydroxybenzoic acids generally have a maximum UV absorption between 240 to 280 nm whereas hydroxycinnamic acids display maxima in the UV spectrum between 300 and 330 nm. Compared to polyphenolics, such as flavonoids, phenolic acids are more water-soluble and more bioavailable in the human body. Hydroxycinnamic acids, such as caffeic and ferulic acid for example, are directly absorbed and circulated into the bloodstream 17.
OH
O
Benzoic acid Cinnamic acid
OH
O
Figure 2.3 Chemical structures of benzoic acid and cinnamic acid; the primary structures of all